This project deploys a range of structural techniques to examine normal synaptic structure. These approaches have in common their dependence on rapid freezing and direct visualization of living brain by light microscopical techniques. Up until now this project has been engaged in explorations of various live brain preparations suitable for these purposes. Recently, an isolated whole brain preparation maintained in vitro by vascular perfusion as well superfusion with artificial cerebrospinal fluid (CSF) has been evaluated. The ultrastructure of surface samples of isolated brains rapidly excised, quick frozen and freeze substituted served as a benchmark to choose a perfusion fixative yielding realistic images of synaptic structures. It could now be determined to what extent deeper cortical regions of perfused brains remained structurally intact. Throughout 2 hr of perfusion, the morphology of synaptic structures in the isolated brain remained equivalent to the normal brain perfuse~fixed in situ. These results provide an excellent method for structural work on the isolated brain, and show that the isolated brain can be used for studies of synaptic structure depending on rapid freeze fixation, and are in agreement with the reported persistence of electrophysiological functions in this preparation. The comprehensive effort to develop methods for making and maintaining organotypic brain cultures continues (see also Project #Z01 NS 02610~10 LN). Up until now it has proven consistently difficult to maintain large pieces of mature brain in culture conditions, but new approaches to this problem are being evaluated.